The purpose of this project is to develop vaccines against otitis media caused by nontypeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis. In this fiscal year, new efforts have been made to develop peptide vaccines against one of the above organism, NTHi. A major surface-exposed component of NTHi, lipooligosaccharide (LOS), is a virulence factor as well as a potential protective antigen. LOS is too toxic to be administered in humans. However, detoxified LOS is a T cell-independent small molecule and is poorly immunogenic in vivo, so we converted LOS into a nontoxic T cell-dependent antigen through the use of peptides that mimic the LOS by screening a phage display peptide library with a rabbit antibody specific for NTHi LOS. Fifty-six phage clones were found to share LOS mimicry molecules. Among them, 22 clones were subjected to DNA sequencing, and four consensus sequences were identified. Three out of the four synthetic peptides showed strong binding reactivity to the rabbit anti-LOS antibody and also a mouse bactericidal monoclonal anti-LOS antibody in vitro, and elicited specific serum anti-LOS antibodies in rabbits (27 to 81-fold) after conjugation with keyhole limpet hemosyanin. Passive immunization with the rabbit antisera resulted in a significantly enhanced pulmonary bacterial clearance in a mouse model. The enhanced bacterial clearance was eliminated if the rabbit serum was pre-absorbed with NTHi LOS. These data indicate that the peptide mimotopes of LOS that we have identified might be potential components of peptide vaccines against NTHi. Additional important investigations in this fiscal year include to observe if intranasal immunization with a detoxified LOS-tetanus toxoid (dLOS-TT) conjugate vaccine would generate protective immunity against NTHi in a mouse model of nasopharyngeal clearance. The rational is that both NTHi and M. catarrhalis are important human mucosal pathogens that cause otitis media in children and respiratory tract infection in adults. An optimal defense against mucosal pathogens would be mucosal vaccines since they are capable of inducing both mucosal and systemic immunity. Our results demonstrated that intranasal immunization with dLOS-TT plus adjuvant significantly induced LOS-specific IgA antibodies in mouse external secretions, especially in nasal wash (90-fold), bronchoalveolar lavage fluid (25-fold), saliva (13-fold) and fecal extract (3-fold). LOS-specific IgA antibody forming cells were also found in mucosal and lymphoid tissues with their highest numbers in the nasal passage (528 per 106 cells). In addition, the intranasal immunization elicited a significant rise of LOS-specific IgG (32-fold) and IgA (13-fold) in serum. For the immunized mice which had been challenged through the nose with 107 live NTHi strain 9274, the vaccine group showed a significant reduction (74%-77%) of NTHi, compared to that of control groups with CT alone or dLOS plus CT (p<0.05). Negative correlations were found between bacterial counts and the levels of nasal wash IgA or IgG, saliva IgA or serum IgG. The clearance of five heterologous strains was investigated and revealed a significant clearance in strains 3198, 5657 and 7502 but not in strains 1479 and 2019. These data suggest that intranasal immunization with dLOS-TT vaccine elicits both mucosal and systemic immunity against NTHi and enhances bacterial clearance from nasopharynx in mice. Such a vaccine and vaccination regime may be applicable to humans.